Abstract: Self-regulated learning conducted through metacognitive monitoring and scientific inquiry can be influenced by many factors, such as emotions and motivation, and are necessary skills needed to engage in efficient hypothesis testing during game-based learning. Although many studies have investigated metacognitive monitoring and scientific inquiry skills during game-based learning, few studies have investigated how the sequence of behaviors involved during hypothesis testing with game-based learning differ based on both efficiency level and emotions during gameplay. For this study, we analyzed 59 undergraduate students’ (59% female) metacognitive monitoring and hypothesis testing behavior during learning and gameplay with CRYSTAL ISLAND, a game-based learning environment that teaches students about microbiology. Specifically, we used sequential pattern mining and differential sequence mining to determine if there were sequences of hypothesis testing behaviors and to determine if the frequencies of occurrence of these sequences differed between high or low levels of efficiency at finishing the game and high or low levels of facial expressions of emotions during gameplay. Results revealed that students with low levels of efficiency and high levels of facial expressions of emotions had the most sequences of testing behaviors overall, specifically engaging in more sequences that were indicative of less strategic hypothesis testing behavior than the other students, where students who were more efficient with both levels of emotions demonstrated strategic testing behavior. These results have implications for the strengths of using educational data mining techniques for determining the processes underlying patterns of engaging in self-regulated learning conducted through hypothesis testing as they unfold over time; for training students on how to engage in the self-regulation, scientific inquiry, and emotion regulation processes that can result in efficient gameplay; and for developing adaptive game-based learning environments that foster effective and efficient self-regulation and scientific inquiry during learning.